Sensitive and rapid quantification of C-reactive protein using quantum dot-labeled microplate immunoassay

Abstract

Background: High-sensitivity C-reactive protein (hs-CRP) assay is of great clinical importance in predicting risks associated with coronary heart disease. Existing hs-CRP assays either require complex operation or have low throughput and cannot be routinely implemented in rural settings due to limited laboratory resources.

Methods: We developed a novel hs-CRP assay capable of simultaneously quantifying over 90 clinical samples by using quantum dots-labeled immunoassay within a standard 96-well microplate. The specificity of the assay was enhanced by adopting two monoclonal antibodies (mAbs) that target distinct hs-CRP epitopes, serving as the coating antibody and the detection antibody, respectively. In the presence of hs-CRP antigen, the fluorescence intensity of the mAb-Ag-mAb sandwich complex captured on the microplate can be read out using a microplate reader.

Results: The proposed hs-CRP assay provides a wide analytical range of 0.001-100 mg/L with a detection limit of 0.06 (0.19) μg/L within 1.5 h. The accuracy of the proposed assay has been confirmed for low coefficient of variations (CVs), 2.27% (intra-assay) and 8.52% (inter-assay), together with recoveries of 96.7-104.2%. Bland-Altman plots of 104 clinical samples exhibited good consistency among the proposed assay, commercial high-sensitivity ELISA, and nephelometry, indicating the prospects of the newly developed hs-CRP assay as an alternative to existing hs-CRP assays.

Conclusion: The developed assay meets the needs of the rapid, sensitive and high-throughput determination of hs-CRP levels within a short time using minimal resources. In addition, the developed assay can also be used to detect and quantify other diagnostic biomarkers by immobilizing specific monoclonal antibodies.

Figure 1

Scheme for the two formats of QL-MI. These assays share the same procedure at the first two steps, including monoclonal antibody coating and antigen introduction. The difference is that A), the QD-mAb conjugate was introduced into the microplate prior to fluorescence detection in the one-step format while B), the QD-streptavidin was added 20 min later than the biotinylated secondary antibody in two-step format.

Figure 2

Influence of storage time (A), immobilization concentration (B), incubation time (C), and detection antibody concentration (D) on the fluorescence intensity. (A) Fluorescence intensity after different storage times (1-10 d) for the QD-streptavidin stock solution only (black line), QD-mAb stock solution only (red line), and 10 mg/L of the purified CRP standard using QD-mAb (green line). (B) Various coating concentrations (1-10 mg/L) of primary anti-CRP monoclonal antibody were employed, and 10 mg/L of serum hs-CRP was introduced into the detection well to judge the highest fluorescence intensity. (C) In each determination, the serum hs-CRP (10 mg/L) was incubated with 4 mg/L of coating antibody for different time periods (varied from 5-50 min with a 5-min interval) to determine the optimal reaction time. (D) Various coating concentrations (0.1-1 mg/L) of capture antibody were introduced into the microplate (4 mg/L coating antibody) and reacted with 10 mg/L serum CRP. Error bars indicate the standard errors of 3 independent experiments.

Figure 3

Calibration curves for hs-CRP detection; 0.001, 0.01, 0.1, 1, 10, and 100 mg/L standard CRP was diluted from CRP stock solution and reacted with optimal capture antibody (4 mg/L) to perform this calibration. A) The best fit for the calibration curve is y = 0.51× + 2.63 with r ² = 0.991 in the one-step format. The black solid round is the fluorescence intensities of individual hs-CRP concentrations and the red straight line is the fitted curve. B) The best fit for the calibration curve is y = 0.49× + 2.82 with r² = 0.994 in the two-step format. The black solid square represents the fluorescence intensities of individual hs-CRP concentrations with the red straight line representing the fitted curve. Error bars indicate the standard errors of 3 independent experiments.

Figure 4

Cross-reaction of the two assay formats; 100 μL of a 10 mg/L solution of albumin (red column), hemoglobin (green column), and immunoglobulin G (blue column) were introduced into the coating antibody-immobilized microplate to perform the cross-reaction. No significant differences were observed between the fluorescence intensities induced by albumin, hemoglobin, and immunoglobulin G. However, the fluorescence intensities induced by nonspecific binding were significantly lower than those induced by the specific hs-CRP antigen in the one-step assay (p < 0.05) and in the two-step assay (p < 0.05). Error bars indicate the standard errors of 3 independent experiments.

Figure 5

Comparison of results for 104 clinical samples from the proposed QL-MI immunoassay, ELISA, and nephelometry. Each sample was detected for three duplicates, and the mean of three results was taken for comparison. Black circles represent the comparison of 104 clinical samples between the one-step QL-MI and hs-ELISA (A); two-step QL-MI and hs-ELISA (B); one-step QL-MI and nephelometry (C); and two-step QL-MI and nephelometry (D). The black solid line represents the mean difference; dashed lines represent mean difference ± 1.96 times of the SD of the differences.